Development of expression systems for the production of recombinant proteins is important for providing a source of a given protein for research or therapeutic use. Expression systems have been developed for both prokaryotic cells such as E. coli, and for eukaryotic cells, which include both yeast and mammalian cells. Expression in mammalian cells, for example Chinese hamster ovary (or xe2x80x9cCHOxe2x80x9d) cells, is often preferred for the manufacture of therapeutic proteins, since post-translational modifications in such expression systems are more likely to resemble those found in human cells expressing proteins than the type of post-translational modifications that occur in microbial (prokaryotic) expression systems.
Transcription of eukaryotic genes is regulated by a variety of cis- and trans-acting regulatory elements. (Dillon et.al.,(1993) Trends Genet. 9:134). Two of the best characterized cis- elements are promoters and enhancers. Promoters are DNA sequences immediately 5xe2x80x2 to the coding sequence of the gene and encompass multiple binding sites for trans-acting transcription factors, forming the basic transcription apparatus. Enhancers are also composed of multiple binding sites for trans-acting transcription factors but can be found far upstream or downstream of coding sequences or even within introns. These elements can also act in an orientation independent manner. Activities of promoters and enhancers can be detected in transient expression systems and they contain elements which may or may not be tissue specific.
Another category of cis- acting regulatory elements are ones which are believed to regulate the chromatin structure including locus control regions (xe2x80x9cLCRsxe2x80x9d) (Grosveld et.al., (1987) Cell 51:975), matrix attachment regions (xe2x80x9cMARsxe2x80x9d) (Phi-Van et.al., (1990) Mol. Cell. Biol. 10:2302), scaffold attachment regions (xe2x80x9cSARsxe2x80x9d) (Gasser and Laemmli (1987) Trens Genet. 3:16), insulator elements (Kellum and Schedl (1991) Cell 64:941) and Nuclear matrix-Associating DNAs (Bode J et.al., (1992) Science 255:195). MAR""s and SAR""s are similar to enhancers in that they are able to act over long distances, but are unique in that their effects are only detectable in stably transformed cell lines or transgenic animals. LCRs are also dissimilar to enhancers in that they are position and orientation dependent, and are active in a tissue specific manner.
Recombinant expression plasmids comprising a gene of interest that encodes all or a portion of a desired protein are routinely used to generate stable CHO cells or transfectomas, expressing the desired recombinant protein. These recombinant plasmids randomly integrate into the genome of the host producing recombinant proteins. However, the frequency of transfectomas carrying the stably integrated recombinant gene that are capable of expressing a desired recombinant protein at high levels is quite low. Usually a large number of stably transfected mammalian transfectomas must be screened to identify clones which express the recombinant proteins at high levels. This is widely believed to be due to the effects of the genomic environment (hot spot) and the plasmid copy number, especially in light of the large size of the mammalian genome and the fact that only 0.1% of the genomic DNA contains transcriptionally active sequences. (Little (1993) Nature 366:204). It is highly unlikely that the current technology of random plasmid integration into the genome of CHO cells will result in the insertion of a recombinant gene into a transcriptional hot spot that is capable of gene amplification leading to high levels of gene expression.
Expression augmenting sequences have been disclosed to increase expression of recombinant protein (Morris, A., et.al., Expression augmenting elements (EASE) for eukaryotic expression systems: WO 97/25420). An increase in the frequency of high-level recombinant gene expressing cell lines would provide a much greater pool of high protein expressing transfectomas to choose from. This task can be accomplished by generating homologous recombinant plasmids targeted to a transcriptional hot spot and devising a means to select for such transfectomas.
Novel transcription regulatory sequences, referred to herein as HIRPE (Hot spot for Increased Recombinant Protein Expression), that facilitate increased expression of recombinant proteins in mammalian host cells, are disclosed. A preferred embodiment of the invention is a HIRPE that was obtained from CHO cell genomic DNA.
The present invention discloses a HIRPE from a genomic locus in the CHO genome that is capable of high recombinant gene expression. These loci contain sequence elements that define origin of replication, gene amplification, and MARs in the mammalian genome. In a most preferred embodiment of the invention, the HIRPE is selected from the group consisting of (a) DNAs comprising nucleotides 1 through 5039 of SEQ ID NO:1; (b) fragments of SEQ ID NO:1 that have HIRPE activity; (c) nucleotide sequences complementary to (a) and/or (b); (d) nucleotide sequences that are at least about 80%, more preferably about 90%, and more preferably about 95% identical in nucleotide sequence to (a), (b) and/or (c) and that exhibit HIRPE activity; and (e) combinations of the foregoing nucleic acid sequences that exhibit HIRPE activity.
Expression vectors comprising the novel HIRPE are able to transform CHO cells to increase expression of recombinant proteins. Thus, another embodiment of the invention is an expression vector comprising a HIRPE. In a preferred embodiment, the expression vector further comprises a eukaryotic promoter/enhancer driving the expression of all or a portion of a protein of interest. Two or more expression vectors may be used to transfect a cell (e.g., CHO cell), wherein each vector comprises a nucleic acid sequence encoding different polypeptides that assemble (when expressed) to form a desired protein. In a more preferred embodiment, the expression vector comprises a plasmid wherein a first exon encodes a gene of interest and a second exon encodes an amplifiable dominant selectable marker. A preferred marker is dihydrofolate reductase (xe2x80x9cDHFRxe2x80x9d); other amplifiable markers are also suitable for use in the inventive expression vectors.
Mamnmalian host cells can be transformed with an expression vector of the present invention to produce high levels of recombinant protein. Accordingly, another embodiment of the invention provides a mammalian host cell transformed with an expression vector of the present invention. Also within the scope of the present invention are mammalian host cells transformed with two expression vectors, wherein each of said two expression vectors encodes a polypeptide subunit that when coexpressed assembles into a desired protein with biological activity. In a most preferred embodiment, the host cells are CHO cells.
The invention also provides a method for obtaining a recombinant protein, comprising transforming a host cell with an expression vector of the present invention, culturing the transformed host cell under conditions promoting expression of the protein, and recovering the protein. In a preferred application of the invention, transformed host cells are selected with two selection steps, the first to select for cells expressing the dominant amplifiable marker, and the second step for high expression levels and/or amplification of the marker gene as well as the gene of interest. In a preferred embodiment, the recombinant protein comprises CTLA4-Ig or a variant thereof, such as substitution, addition and deletion variants to the amino acid sequence to produce different forms of a CTLA4-Ig molecule. . For an extensive review of CTLA4 and CTLA4 fusion proteins (e.g., CTLA4-Ig) see WO 93/00431 incorporated herein in its entirety by reference.